/ . Embryol. exp. Morph. Vol. 55, 247-256, pp. 1980
Printed in Great Britain © Company of Biologists Limited 1980
247
Studies on the female-sterile phenotype
of l(l)su(f)ts76a, a temperature-sensitive allele of
the suppressor of forked mutation in
Drosophila melanogaster
BY THOMAS G. WILSON 1
From the Department of Biological Sciences, Northwestern University,
Evanston, Illinois
SUMMARY
A new allele of the suppressor of forked [su(f)] mutation in Drosophila melanogaster has
been found and designated l(l)su(f)ts70a. It is temperature-sensitive for suppression of forked
(/) and has additional temperature-sensitive phenotypes of lethality, female sterility, and
abnormal bristle formation at 29 °C. It closely resembles two other conditional alleles of
su(f), l{l)su(f)lsG7g and I{l)ts726. Female sterility at 29 °C is characterized by both disorganized egg chambers in the ovarioles and also chorion-deficient oocytes. Both of these
abnormalities may be the result of premature follicle cell death. The observations on l{l)su(f)
ts76a a r e cons i s tent with the proposal that the similar allele, l(J)ts726, is a cell-lethal mutation specifically affecting mitotically active cells.
INTRODUCTION
Numerous loci have been described for Drosophila melanogaster which express
phenotypes of lethality (see Lindsley & Grell, 1968) or female sterility (King,
1970). Analysis of these loci is considerably easier when temperature-sensitive
(ts) alleles are found (Suzuki, 1970), allowing one to determine sensitive periods
and resulting phenotypes simply by shifting animals to the restrictive temperature. In a screen for ts juvenile hormone null mutants, I selected for phenotypes expected of a juvenile hormone deficient mutant, ts larval-lethality and
non-vitellogenic female sterility (Vogt, 1943; Postlethwait & Weiser, 1973). I
recovered one larval-lethal, female-sterile mutant which proved to be an allele
of suppressor of forked [su(f)]. (Subsequent characterization of this allele
demonstrated ample vitellogenesis and thus the unlikelihood of a juvenile
hormone deficiency.) Two other labs have reported similar su(f) alleles (Dudick,
Wright & Brothers, 1974; Russell, 1974). Both of these mutants possess an array
of ts phenotypes, including suppression of the forked (/) mutation, female
1
Present address: Department of Zoology, University of Vermont, Burlington, Vermont
05405, U.S.A.
248
T. G. WILSON
sterility and lethality. In addition, Russell's mutant, I(l)ts726, shows abnormal pattern formation in leg and eye-antennai imaginal disc tissue when
larvae are subjected to a restrictive temperature pulse, and bristle deficiencies
when pupae are shifted to the higher temperature. It has been postulated that
this phenotype results from imaginal disc cell death, and I{l)ts726 has proven
useful in examining pattern formation in Drosophila (Russell, 1974; Clark &
Russell, 1977; Russell, Girton & Morgan, 1977).
The ts suppression and lethality phenotypes of this new allele, J(l)su(f)ts7Ga,
are very similar to those described for the other ts alleles and will be only mentioned in this paper. The female-sterility phenotype will be described in detail.
The results from these studies support the hypothesis of Russell (1974) that
I(l)ts726 is a cell-death mutant specific for cells undergoing mitosis.
MATERIALS AND METHODS
Stocks
The Oregon-R wild-type stock has been maintained as an inbred line at Northwestern University for 20 years. The FM7, y31dv B balancer stock (see Lindsley
& Grell, 1968, for stock description) was obtained from the Oak Ridge collection.
All other stocks were obtained from the Bowling Green collection. The y, cv, v
and / genes in the y cv vf ts76a chromosome were derived from a y cv vf car
chromosome from that stock. The l(l)su(f)ts67° stock used for allele complementation tests also contained l(l)mystsZ. In this report the genotypes of
heterozygotes have the chromosome of maternal origin written first.
Mutagenesis
Only X chromosomes were screened in this work because of the ease of stock
manipulations relative to screening autosomes. Male Oregon-R flies were fed
ethyl methane sulphonate by the procedure of Lewis & Bacher (1968) and
mated with attached-X females of the constitution C(1)DX, yf/Y. F x progeny
were raised at 23 °C and males singly mated to C(1)DX females. A sample
culture of F 2 progeny from each single-pair mating was raised at 29 °C to test
for pre-adult lethality, indicated by the absence of F 2 adult males. Homozygous
females were constructed via an FM7 balancer chromosome from each culture
exhibiting a pre-adult-lethality phenotype. Such females were tested for ts
sterility by shifting to 29 °C at eclosion and monitoring fertility over a period of
several days.
Staging for temperature shifts
Temperature-shift experiments were carried out as described by Suzuki (1970).
For the larval temperature-shift experiments larvae were isolated from 0-1 h
after hatching at 24°(±1 °C) and either placed at 29°(±1 °C) (shift-down
Female-sterile phenotype of l(l)su(f)ts76a in Drosophila
249
Fig. 1. Scanning electron micrographs of tergites of (a) ts76a/+ and (b) ts76a/ts76a
shifted to 29 °C at pupariation for 40 h. x 200.
experiments) or maintained at 24 °C (shift-up experiments) for various lengths
of time before shifting to the lower or higher temperature. Similar shifts were
made for pupae of different ages after they were restaged at the white puparial
stage (Bodenstein, 1950).
Miscellaneous
Fecundity was determined from daily egg collections from females at 24 °C
placed 20 per shell vial together with 20 Oregon-R males. Fertilized eggs were
determined to be those turning brown within 3 days after oviposition (Wright,
1973); unfertilized eggs remain white during this time. Ovary transplants were
done using the methods outlined by Ursprung (1967). Decisions regarding ovary
autonomy were made after examination of at least 12 transplants for each
experimental condition. Feulgen-stained whole mounts (King, Rubinson &
Smith, 1956) of ovaries were prepared for examination. Flies were raised on a
standard agar-cornmeal-yeast diet.
RESULTS
Mutagenesis and mapping
Several ts larval-lethal female-sterile mutants were picked up in the mutagenesis screen. Only one is described in this paper and was designated TSl in the
preliminary work.
TSl was mapped by recombination by crossing TSl males to y cv vfcar females.
Fx females were mated to Oregon-R males, and the F 2 generation was raised at
29 °C. Since preliminary tests had shown TSl hemizygotes to be consistently
100% larval-lethal at 29 °C, all F 2 males carrying the TSl gene were eliminated.
A preliminary examination of 316 surviving F 2 males located TSl 3 map units
250
T. G. WILSON
10
Time (days)
Fig. 2. Fecundity of ts76a females after shifting to 29 °C (solid arrow) and after returning to 24 °C (dashed arrow). The top curve represents the percentage hatch of
morphologically normal eggs laid by these females. Data from 80 females, 20 per shell
vial. Error bars designate ± 1 standard error.
to the right of car. This map position of 65-66 places TSl near su(f), given as
65.9 (Lindsley & Grell, 1968). Since alleles of su(f) have been described which
show lethality and female sterility (Dudick et ah 1974; Russell, 1974), the possibility that TSl is an allele of su{f) presented itself. Allelism to su(f) was tested
by crossing TSl with an allele of su(f), lifysuify867", which also exhibits conditional lethality and female sterility (Dudick et a!. 1974). The
TSl/l(l)su(f)
tsG7g progeny demonstrated ts larval-lethal and female sterile phenotypes indistinguishable from TSl homozygotes. The final proof that TSl is an allele of
su(f) rests on its ability to suppress/. TSl was found to suppress/in a ts manner
analogous to that described for l(l)su(f)ts67g (Dudick et ah 1974). It was concluded that TSl is allelic to su(f) and will be redesignated l(l)su(f)ts76a and
ts76a in this paper.
Pre-adult temperature sensitivity
The temperature-sensitive periods for lethality during development of ts76a
were determined by temperature shift experiments. A period of time extending
from second larval instar until 12 h after pupariation delineates the ts period.
Female-sterile phenotype of l(l)su(f)ts76a in Drosophila
251
m
(b)
0-1 mm
Fig. 3. Photomicrograph of (a) ts76a/+ and (b) ts76a/ts76a ovarioles after 5 days at
29 °C. Note the absence of follicle cells (arrow) in (b).
Dudick et al. (1974) and Russell (1974) found a similar sensitive period in development for their mutants.
Pupae of ts76a shifted to 29 °C within 5 h after pupariation usually failed to
survive eclosion, but those that did survive showed a striking bristle-deficiency
phenotype (Fig. 1). The absence of bristles is more pronounced on abdominal
tergites than on the head and thorax. Russell et al. (1977) also noted defective
bristle formation when I(l)ts726 mid-third instar larvae were shifted to 29 °C.
Female sterility
ts76a females maintained at 24 °C exhibited excellent fertility and fecundity.
When shifted to 29 °C, however, females ceased to oviposit within a 4- to 5-day
period (Fig. 2). During this period, different types of eggs were laid. For the
first two days, the eggs appeared morphologically normal, although egg hatch
decreased after the first day (Fig. 2). Since 70-80% of the unhatched eggs were
fertilized, the decrease in egg hatch was not due to decreasing fertilization. Eggs
laid between two and four days at 29 °C were of two types: (1) morphologically
normal eggs of decreasing hatch in proportion to time of exposure of females to
29 °C; and (2) abnormal eggs, which lacked a chorion, were smaller than normal
and misshapen, and never hatched. After 4 days at 29 °C, 90% of eggs laid were
abnormal; after 5 to 6 days, oviposition ceased. When ts76a females at 29 °C
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T. G. WILSON
were returned to 24 °C, they began laying fertile eggs after 4 days (Fig. 2). The
first few eggs laid were abnormal, but subsequent eggs were morphologically
normal.
Appearance of ovaries
Ovaries of ts76a females were examined as Feulgen-stained whole mounts.
Whole mounts of ovaries from females maintained at 24 °C were morphologically indistinguishable from those of Oregon-R females (see King, 1970, for
a description of oogenesis in Drosophila melanogaster). However, ovaries
from ts76a females shifted to 29 °C within 4 h after eclosion and maintained at
that temperature for 5 days were very different (Fig. 3). Usually, one or two
stage-14 (mature) oocytes were present. Also, several fully vitellogenic (resembling stage-13) oocytes were present in each ovary; these oocytes resembled
the abnormal eggs laid after several days at 29 °C and probably were laid as
abnormal eggs when females were returned to 25 °C. No stage-8 to -11 oocytes
(partially vitellogenic) were seen, suggesting that vitellogenic uptake was not
actively occurring after 5 days at 29 °C, although partially vitellogenic oocytes
have been observed after 3 days at 29 °C. No distinct egg chambers were present;
rather, the vitellaria appeared to be packed haphazardly with cells resembling
nurse cells. Follicle cells were absent, although debris suggestive of dead follicle
cells surrounded late-stage oocytes.
Ovary transplants
To determine if ts76a ovaries were abnormal at 29 °C due to some environmental factor (non-autonomous development) or to a factor within the ovary
(autonomous development), ovary transplants from ts76a into Oregon-R hosts
were performed. ts76a females were raised at 24 °C and ovaries were extirpated
0-4 h after eclosion and transplanted into either of two types of Oregon-R hosts:
2-4 h post-eclosion females or females which had been shifted to 29 °C at
eclosion and maintained at 29 °C for 5 days. All hosts were placed at 29 °C 1 h
after surgery, and host and donor ovaries were examined after 5 days at 29 °C as
Feulgen-stained whole mounts. Host ovaries showed normal development, the
ovaries morphologically indistinguishable from Oregon-R unoperated control
females. Implanted ts76a ovaries appeared similar to those of unoperated ts76a
females shifted to 29 °C 5 days before examination. Therefore, both host and
implant ovaries developed autonomously. Likewise, when ovaries were dissected
from 0-2 h post-eclosion Oregon-R females, and transplanted into ts76a females
which had been shifted to 29 °C at 30 h after pupariation and maintained at that
temperature for 5 days, both donor and host ovaries showed autonomous
development at 29 °C 5 days after implantation.
Male sterility
In order to distinguish between the possibility that ts76a affects oocyte development only or affects gamete development in both sexes, it was important to
Female-sterile phenotype of l(l)su(f)ts76a in Drosophila
253
Time (Days)
Fig. 4. Fertility of males after shifting to 29 °C (solid arrow) and returning to 24 °C
(dashed arrow), O, Oregon-R; • , y cv vfts76a/y+ Y; D, ts 76a. Each curve was
constructed from data from 25 males, one per shell vial with two Oregon-R virgin
females. Females were replaced daily and observed for progeny over a 10-day period
at 24 °C. A male was scored as fertile on a particular day if progeny were produced
from one of the females.
determine if ts76a males become sterile at 29 °C. This experiment was complicated by the observation that Oregon-R males became sterile after 7 days at
29 °C, although the effect was reversible (Fig. 4). When ts76a males were shifted
to 29 °C, they rapidly became irreversibly sterilized (Fig. 4). This effect could
be due to the ts76a mutation or to another EMS-induced mutation on the X
chromosome affecting male sterility. To distinguish between these possibilities
ts76a males were tested whose X chromosome had been largely replaced by a
y cv v/chromosome through recombination, although an undetermined amount
of the original X chromosome between/(56.7 map position) and ts76a (65.9
map position) remained. These y cv v f ts76a males proved to be considerably
more fertile at 29 °C than ts76a males (Fig. 4), presumably due to the elimination
of a mutation affecting male fertility at 29 °C. Thus the results suggest that
ts76a depresses male fertility but does not render males completely sterile at
29 °C. Russell (1974) reported that 29 °C has no adverse effect on male fertility
of I(l)ts726,
DISCUSSION
It is noteworthy that ts alleles of su(f) have been selected in three different
mutant screens. This is due not only to the several phenotypes exhibited by these
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EMB
55
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T. G. WILSON
alleles, but also to the fact that they are highly penetrant and produce such
striking phenotypes. All three alleles have similar phenotypic characteristics.
Cell death was found in imaginal disc and brain tissue of I(l)ts726 by Russell
(1974), and he suggests that it is a cell-death mutant. Subsequently, a histological examination of heat-shocked l(T)ts726 larvae by Clark & Russell (1977)
demonstrated lysosomal activity indicative of cell death in imaginal disc cells. The
molecular basis of the cell death may be defective ribosomal protein, suggested
as the su(f) gene product (Finnerty et al. 1973; Wright, 1973; Dudick et ah
1974 (but see Lambertson, 1976; Vaslet and Berger, 1976).
Russell noted that cell death in l(T)ts726 is somewhat specific, affecting cells
which are actively undergoing mitosis. In addition to imaginal disc cells, affected
cell types include brain cells and abdominal histoblasts (Russell, 1974). With
the finding that follicle cells are sensitive to 29 °C, the present work is in agreement with Russell's results, since follicle cells undergo mitosis during oocyte
development (see King, 1970). The female sterility of ts76& may be due entirely
to premature follicle-cell death. If follicle cells surrounding egg chambers are
responsible for shaping these chambers, then the absence of viable follicle cells
would explain the amorphic arrangement of nurse cells and oocytes in the ovarioles of females shifted to 29 °C. Since follicle cells are responsible for deposition
of the chorion (King & Koch, 1963; Quattropani & Anderson, 1969; Petri,
Wyman & Kafatos 1976), premature death would account for the development of oocytes lacking chorions in females which have been shifted to 29 °C
for several days. At least 25 h at 29 °C is necessary to either kill follicle cells or
impair their functioning. This is evidenced by the observation that ts76a females
shifted to 29 °C at eclosion contain one or two stage-14 oocytes (having chorionic
appendages) when examined 5 days later, as do ovaries transplanted from newly
eclosed ts76a females to Oregon-R hosts which are then shifted to 29 °C.
Approximately 30-35 h are required for newly eclosed wild-type females to form
stage-14 oocytes at 25 °C (calculated from data of Handler & Postlethwait, 1978
and David & Merle, 1968), and probably less time (perhaps 25-30 h) is required for stage-14 oocyte maturation in ts76a females at 29 °C. Therefore,
follicle cells of ts76a females at 29 °C are functional for at least this time period.
Bristle and socket formation is a product of two specialized epidermal cells,
the trichogen and the tormogen, respectively (Lees & Waddington, 1942).
The final differentiative divisions producing these cells occur during a period
of from 14| to 36 h after pupariation, based on the sensitivity of the dividing
cells to X-ray (Poodry, 1975). These cell types are sensitive to 29 °C in ts76a,
since both bristle and socket structures are absent on abdomens of adults shifted
to 29 °C shortly after pupariation. The radiosensitivity period determined by
Poodry for the abdominal bristles allows an estimation of the time required for
the effect of the restrictive temperature on ts76a pupae. Since the radiosensitive
period of abdominal bristles is 24 h after pupariation (Poodry, 1975), and since
the ts period for abdominal bristle formation is 0-5 h after pupariation of ts76a,
Female-sterile phenotype of l(l)su(f) ts76a in Drosophila
255
a minimum of 20 h at 29 °C is required to suppress abdominal bristle and socket
formation. Of course, this calculation assumes that the effect of ts76a is on the
final cell division and not on bristle and socket formation.
Mitosis in Drosophila embryos is a very active process, yet none of the ts
su(f) alleles shows a sensitive embryonic period. This may be due to the presence
of sufficient su(f)+ gene product deposited in the oocyte to satisfy any mitotic
requirements for this product during embryogenesis. The decreasing egg hatch
of morphologically normal, fertilized eggs laid by ts76a females placed at 29 °C
(Fig. 2) possibly reflects insufficient su(f)+ product placed in the oocytes to
satisfy mitotic requirements during embryogenesis.
Other cell types undergoing mitosis are the stem cells of the reproductive
tissues. The fact that female sterility is reversible (Fig. 2) and male sterility is
partially reversible (Fig. 4) suggests that stem cells may not be killed by the
restrictive temperature. The basis for this sensitivity difference between stem cells
and other dividing cell types to ts76a remains to be elucidated.
Russell's laboratory has made extensive use of I{l)ts726 for studying various
aspects of pattern formation. With the finding that ts76a affects follicle cell
viability, perhaps this mutant will be useful for investigating follicle cell functions
during oocyte maturation. One fruitful approach might be an examination of
ovaries of ts76a/+ females shifted to 29 °C after inducing ts76a follicle cell
clones by mitotic recombination.
The author would like to thank Dr Lawrence I. Gilbert for his support, and Drs Robert C.
King and Jack Girton for their helpful comments during this work.
Research supported by National Institutes of Health National Research Service Award
ES 05050-01 to T. G. Wilson, and National Institutes of Health Grant AM 02818 and
National Science Foundation Grant PCM 76-03620 to L. I. Gilbert.
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